Excipient stabilization of polypeptides treated with organic solvents

ABSTRACT

Methods and compositions for excipient stabilization of dry or aqueous polypeptides treated with organic solvents are disclosed, wherein the polypeptide is admixed with a polyol having a molecular weight less than about 70,000 kD.

This is a continuation of application U.S. Ser. No. 08/256,187, filedApr. 8, 1994, now U.S. Pat. No. 5,589,167, which is a 371 of U.S. Ser.No. PCT/US94/01666 filed on 17 Feb. 1994, which is a CIP of U.S. Ser.No. 08/021,421, filed Feb. 23, 1993, now abandoned.

FIELD OF THE INVENTION

This invention relates to the use of excipients to stabilize both dryand aqueous formulations of polypeptides treated with organic solvents.

BACKGROUND OF THE INVENTION

Pharmaceutical preparations of polypeptides are sensitive todenaturation and degradation upon formulation and storage. Polyols havebeen used to stabilize proteins and other macromolecules in aqueousformulations and in air drying or lyophilization from aqueous solutions.

U.S. Pat. No. 4,297,344 discloses stabilization of coagulation factorsII and VIII, antithrombin III, and plasminogen against heat by addingselected amino acids such as glycine, alanine, hydroxyproline,glutamine, and aminobutyric acid, and a carbohydrate such as amonosaccharide, an oligosaccharide, or a sugar alcohol.

European Patent Application Publication No. 0 303 746 disclosesstabilization of growth promoting hormones with polyols consisting ofnon-reducing sugars, sugar alcohols, sugar acids, pentaerythritol,lactose, water-soluble dextrans, and Ficoll, amino acids, polymers ofamino acids having a charged side group at physiological pH, and cholinesalts.

European Patent Application Publication No. 0 193 917 discloses abiologically active composition for slow release characterized by awater solution of a complex between a protein and a carbohydrate.

Australian Patent Application No. AU-A-30771/89 discloses stabilizationof growth hormone using glycine and mannitol.

U.S. Pat. No. 5,096,885 discloses a formulation of hGH forlyophilization containing glycine, mannitol, a non-ionic surfactant, anda buffer.

The use of polyethylene glycols to stabilize proteins is reviewed inPharm Res. 8:285-291, 1991.

Examples of the use of trehalose and other polyols for the stabilizationof proteins during drying in aqueous systems include the following.

U.S. Pat. No. 4,891,319 discloses the preservation of sensitive proteinsand other macromolecules in aqueous systems by drying at ambienttemperatures and at atmospheric pressure in the presence of trehalose.

U.S. Pat. No. 5,149,653 discloses a method of preserving live viruses inan aqueous system by drying in a frozen state or at ambient temperature,in the presence of trehalose.

Polyols have also been used to stabilize dry drug formulations as, forexample, in WO 8903671, filed May 5, 1989, which discloses the additionof a stabilizer such a gelatin, albumin, dextran, or trehalose to amixture of a finely powdered drug suspended in a oily medium.

Treatment of a polypeptide with an organic solvent such as methylenechloride poses the problem of denaturation of the polypeptide ofinterest. Thus, it is an object of this invention to provide a methodfor stabilizing polypeptides in aqueous formulations treated withorganic solvents.

It is another object of the invention to stabilize dry polypeptidestreated with organic solvents.

It is another object of the invention to provide a method forstabilization of encapsulated polypeptides.

It is another object of the invention to provide a polypeptidestabilized by an excipient for use in a controlled release formulation,wherein the polypeptide is treated with an organic solvent.

SUMMARY OF THE INVENTION

One aspect of the invention is a method of stabilizing a polypeptideagainst denaturation when treated with an organic solvent, wherein themethod comprises admixing the polypeptide with a polyol, wherein themolecular weight of the polyol is less than about 70,000 kD.

Another aspect of the invention is a method of formulating a polypeptidecomprising

a) admixing the polypeptide in an aqueous solution with a polyol havinga molecular weight less than about 70,000 kD; and

b) treating the polypeptide in the aqueous solution with an organicsolvent.

Another aspect of the invention is a method of formulating a drypolypeptide for controlled release comprising

a) admixing the polypeptide with an excipient, wherein said excipient isa polyol having a molecular weight less than about 70,000 kD; and

b) treating the product of step a) with an organic solvent.

Another aspect of the invention is a composition for controlled releaseof a polypeptide comprising a polypeptide admixed with an excipient, theexcipient being a polyol having a molecular weight less than about70,000 kD, wherein the polypeptide admixed with the excipient is treatedwith an organic solvent and is encapsulated in a polymer matrix.

DETAILED DESCRIPTION OF THE INVENTION

A. DEFINITIONS

The term "polyol" as used herein denotes a hydrocarbon including atleast two hydroxyls bonded to carbon atoms. Polyols may include otherfunctional groups. Examples of polyols useful for practicing the instantinvention include sugar alcohols such as mannitol and trehalose, andpolyethers.

The term "polyether" as used herein denotes a hydrocarbon containing atleast three ether bonds. Polyethers may include other functional groups.Polyethers useful for practicing the invention include polyethyleneglycol (PEG).

The term "dry polypeptide" as used herein denotes a polypeptide whichhas been subjected to a drying procedure such as lyophilization suchthat at least 50% of moisture has been removed.

The term "encapsulation" as used herein denotes a method for formulatinga therapeutic agent such as a polypeptide into a composition useful forcontrolled release of the therapeutic agent. Examples of encapsulatingmaterials useful in the instant invention include polymers or copolymersof lactic and glycolic acids, or mixtures of such polymers and/orcopolymers, commonly referred to as "polylactides."

The term "admixing" as used herein denotes the addition of an excipientto a polypeptide of interest, such as by mixing of dry reagents ormixing of a dry reagent with a reagent in solution or suspension, ormixing of aqueous formulations of reagents.

The term "excipient" as used herein denotes a non-therapeutic agentadded to a pharmaceutical composition to provide a desired consistencyor stabilizing effect.

The term "organic solvent" as used herein is intended to mean a carboncompound containing solvent. Exemplary organic solvents includemethylene chloride, ethyl acetate, dimethyl sulfoxide, tetrahydrofuran,dimethylformamide, and ethanol.

"Treating" a polypeptide with an organic solvent as used herein refersto mixing a dry polypeptide with an organic solvent, or making anemulsion of a polypeptide in an aqueous formulation with an organicsolvent, creating an interface between a polypeptide in an aqueousformulation with an organic solvent, or extracting a polypeptide from anaqueous formulation with an organic solvent.

"Polypeptide" as used herein refers generally to peptides and proteinshaving more than about 10 amino acids.

B. GENERAL METHODS

In general, both aqueous formulations and dry polypeptides may beadmixed with an excipient to provide a stabilizing effect beforetreatment with an organic solvent. An aqueous formulation of apolypeptide may be a polypeptide in suspension or in solution. Typicallyan aqueous formulation of the excipient will be added to an aqueousformulation of the polypeptide, although a dry excipient may be added,and vice-versa. An aqueous formulation of a polypeptide and an excipientmay be also dried by lyophilization or other means. Such driedformulations may be reconstituted into aqueous formulations beforetreatment with an organic solvent.

The excipient used to stabilize the polypeptide of interest willtypically be a polyol of a molecular weight less than about 70,000 kD.Examples of polyols that maybe used include trehalose, mannitol, andpolyethylene glycol. Typically, the mass ratio of trehalose topolypeptide will be 100:1 to 1:100, preferably 1:1 to 1:10, morepreferably 1:3 to 1:4. Typical mass ratios of mannitol to polypeptidewill be 100:1 to 1:100, preferably 1:1 to 1:10, more preferably 1:1 to1:2. Typically, the mass ratio of PEG to polypeptide will be 100:1 to1:100, preferably 1:1 to 1:10. Optimal ratios are chosen on the basis ofan excipient concentration which allows maximum solubility ofpolypeptide with minimum denaturation of the polypeptide.

The formulations of the instant invention may contain a preservative, abuffer or buffers, multiple excipients, such as polyethylene glycol(PEG) in addition to trehalose or mannitol, or a nonionic surfactantsuch as Tween® surfactant. Non-ionic surfactants include a polysorbate,such as polysorbate 20 or 80, etc., and the poloxamers, such aspoloxamer 184 or 188, Pluronic® polyols, and otherethylene/polypropylene block polymers, etc. Amounts effective to providea stable, aqueous formulation will be used, usually in the range of fromabout 0.1% (w/v) to about 30% (w/v).

Buffers include phosphate, Tris, citrate, succinate, acetate, orhistidine buffers. Most advantageously, the buffer is in the range ofabout 2 mM to about 100 mM. Preferred buffers include sodium succinateand potassium phosphate buffers.

Preservatives include phenol, benzyl alcohol, metacresol, methylparaben, propyl paraben, benzalconium chloride, and benzethoniumchloride. The preferred preservatives are 0.2-0.4% (w/v) phenol and0.7-1% (w/v) benzyl alcohol, although the type of preservative and theconcentration range are not critical.

In general, the formulations of the subject invention may contain othercomponents in amounts not detracting from the preparation of stableforms and in amounts suitable for effective, safe pharmaceuticaladministration. For example, other pharmaceutically acceptableexcipients well known to those skilled in the art may form a part of thesubject compositions. These include, for example, various bulkingagents, additional buffering agents, chelating agents, antioxidants,cosolvents and the like; specific examples of these could includetrihydroxymethylamine salts ("Tris buffer"), and disodium edetate.

Polypeptides of interest include glycosylated and unglycosylatedpolypeptides, such as growth hormone, the interferons, and viralproteins such as HIV protease and gp120.

The stabilized polypeptide of the instant invention may be formulatedfor sustained release, especially as exposure to organic solvents is acommon step in many of such preparations. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the polypeptide, which matrices are inthe form of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels e.g.,poly(2-hydroxyethylmethacrylate) as described by Langer, et al., J.Biomed. Mater. Res., 15:167-277 (1981) and Langer, Chem. Tech.,12:98-105 (1982) or poly(vinylalcohol)!, polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid and gammaethyl-L-glutamate (Sidman, et al., Biopolymers, 22:547-556 1983!),non-degradable ethylene-vinyl acetate (Langer, et al., supra),degradable lactic acid-glycolic acid copolymers such as the LupronDepot™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid(EP 133,988). While polymers such as ethylene-vinyl acetate and lacticacid-glycolic acid enable release of molecules for over 100 days,certain hydrogels release polypeptides for shorter time periods. Whenencapsulated polypeptides remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for polypeptidestabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S--S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Sustained-release ligand analogs or antibody compositions also includeliposomally entrapped polypeptides. Liposomes containing polypeptidesare prepared by methods known per se: DE 3,218,121; Epstein, et al.,Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); Hwang, et al., Proc.Natl. Acad. Sci. USA, 77:4030-4034 (1980); EP 52,322; EP 36,676; EP88,046; EP 143,949; EP 142,641; Japanese patent application 83-118008;U.S. Pat. No. 4,485,045 and U.S. Pat. No. 4,544,545; and EP 102,324.Ordinarily the liposomes are of the small (about 200-800 Angstroms)unilamelar type in which the lipid content is greater than about 30 mol.% cholesterol, the selected proportion being adjusted for the optimalligand analogs therapy. Liposomes with enhanced circulation time aredisclosed in U.S. Pat. No. 5,013,556.

EXPERIMENTAL EXAMPLES EXAMPLE I Stabilization of Aqueous Formulations

Recombinant human growth hormone (hGH) and recombinant humangamma-interferon (hIFN-γ) were formulated with various excipients foranalysis of the excipient effects on stabilization in the organicsolvent, methylene chloride. Optimal formulations were generally thosethat yielded the maximum soluble polypeptide concentration and thegreatest recovery of native polypeptide after treatment with methylenechloride. The maximum solubility of hGH in each solution was determinedthrough the continuous addition of hGH lyophilized in ammoniumbicarbonate buffer to the solution and the solubility limit was definedas the concentration at which addition of polypeptide resulted inprecipitation. The maximum solubility of hIFN-γ was measured by adding aconcentrated stock solution (264 mg/ml hIFN-γ, 10 mM Na succinate, pH 5)to concentrated excipient solutions. The apparent solubility limit ofhIFN-γ was not observed for any of the formulations at these conditions,but long term storage of the stock solution did result in precipitationas the result of a pH increase (final solution, pH 6). Both polypeptideformulations were tested for stability in methylene chloride by additionof 100 μl of the polypeptide solution to 1 ml of methylene chloride. Themixture was then sonicated for 30 sec. After sonication, the polypeptidewas extracted from the organic phase by dilution into 50 ml ofexcipient-free buffer (50 mM phosphate buffer, pH 8 for hGH; 10 nMsuccinate buffer, pH 5 for hIFN-γ). The amount of soluble polypeptiderecovered was determined by ultraviolet absorbance measurements and theamount of monomeric polypeptide was assessed by size exclusionchromatography.

Both polypeptides were tested for stability with trehalose, mannitol,carboxymethylcellulose (CMC), Tween® 20 surfactant, dextran, gelatin,and polyethylene glycol. Previous studies with hGH indicated thatformulations containing an equal mass ratio of polypeptide and mannitolstabilized the polypeptide from denaturation and provided a maximumsoluble polypeptide concentration of 200 mg/ml (100 mM phosphate, 200mg/ml mannitol, pH 8). Trehalose formulations containing mass ratios Ofexcipient to polypeptide of 1:4 and 1:3 yielded the highestconcentration of soluble polypeptide, 400 mg/ml and 300 mg/ml,respectively. In addition, when the lyophilized polypeptide in theseformulations was treated with methylene chloride, complete recovery ofsoluble monomeric hGH was achieved. hGH formulations containing mannitolor mannitol with PEG resulted in similar recovery of monomeric hGH, butthe mass ratio (excipient/polypeptide) required to prevent denaturationwas greater than that of the trehalose formulations (mannitol: 1:1;mannitol/PEG 1:1 or 1:2; trehalose: 1:3 or 1:4) (Table I). Therefore,trehalose provided high hGH solubility and protection from denaturationin methylene chloride at a lower mass concentration. In the absence ofexcipients, the solubility of hGH was much lower (about 106 mg/ml) andthe polypeptide was more susceptible to denaturation.

                  TABLE I                                                         ______________________________________                                        Methylene chloride testing                                                    of aqueous hGH formulations.                                                                Soluble Monomer                                                               Recovered.sup.b                                                                            Maximum Solubility.sup.c                           Formulation.sup.a                                                                           (mg/ml)      (mg/ml)                                            ______________________________________                                        100 mg/ml PEG 12.2         96.4                                               (3350 MW)                                                                     50 mg/ml PEG  34.7         89.6                                               10 mg/ml PEG  37.2         128.3                                              100 mg/ml Mannitol                                                                          66.0         98.0                                               50 mg/ml Mannitol                                                                           46.2         106                                                10 mg/ml Mannitol                                                                           56.0         106                                                100 mg/ml Dextran 70                                                                        34.6         112.6                                              50 mg/ml Dextran 70                                                                         64.6         146.1                                              10 mg/ml Dextran 70                                                                         38.4         167.1                                              2% CMC        44.7         91.9                                               100 mg/ml Trehalose                                                                         113.9        267.3                                              50 mg/ml Trehalose                                                                          82.8         275                                                10 mg/ml Trehalose                                                                          92.0         267.3                                              4 mg/ml PEG (3350 MW),                                                                      102.6        243.7                                              96 mg/ml Mannitol                                                             10 mg/ml PEG (3350                                                                          104.0        184                                                MW), 90 mg/ml Mannitol                                                        20 mg/ml PEG (3350 MW)                                                                      139.9        240                                                80 mg/ml Mannitol                                                             2% Gelatin    21.9         70.5                                               100 mg/ml PEG 69.2         131.5                                              (1000 MW)                                                                     50 mg/ml PEG  84.3         246.5                                              10 mg/ml PEG  126.5        226.3                                              4 mg/ml PEG (1000 MW)                                                                       122.3        230.3                                              96 mg/ml Trehalose                                                            10 mg/ml PEG (1000 MW)                                                                      58.4         218.7                                              90 mg/ml Trehalose                                                            20 mg/ml PEG (1000 MW)                                                                      75.3         207.5                                              80 mg/ml Trehalose                                                            No excipient  65.0         106.3                                              ______________________________________                                         .sup.a All solutions contained 10 mM NaPO4, pH 8.                             .sup.b Polypeptide extracted out of methylene chloride (fraction of total     as determined by absorbance at 278 nm multiplied by the fraction of           monomer recovered from SECHPLC. Polypeptide treated at maximum solubility     .sup.c Maximum solubility was defined as the maximum amount of                unformulated hGH that would dissolve in each buffer.                     

For studies with hIFN-γ, both mannitol and trehalose were the bestexcipients tested. When mannitol was used at a mass ratio(excipient/polypeptide) of 1:3, the amount of soluble dimer in solution(as determined by size exclusion chromatography) after methylenechloride treatment was equivalent to the amount in the startingmaterial. However, the mannitol formulations yielded less than 60%recovery of total soluble polypeptide. In contrast, the trehaloseformulation with a mass ratio of 1:2.5 gave an 80% recovery of totalsoluble polypeptide and the same fraction of soluble dimer (asdetermined by size exclusion chromatography, denoted native SEC-HPLC) asthe starting material (Table II). Excipient-free polypeptideformulations treated with methylene chloride retained 10% of the initialsoluble dimer (as determined by native SEC-HPLC) after methylenechloride treatment and the total soluble polypeptide recovery was lessthan 60%. When assayed by size exclusion chromatography in 0.2M NaPO₄/0.1% SDS at pH 6.8 (denoted SDS SEC-HPLC), all formulations weregreater than 99% monomer before and after methylene chloride treatment.

For both polypeptides, a dramatically lower recovery of monomericpolypeptide was observed after methylene chloride treatment for allformulations containing Tween® 20 surfactant. Although other surfactantshave not been studied, it is likely that hydrophobic molecules such asTween® 20 surfactant stabilize the denatured polypeptides while sugarssuch as mannitol and trehalose stabilize the native polypeptide.

                  TABLE II                                                        ______________________________________                                        Methylene chloride testing                                                    of aqueous hIFN-γ formulations                                                        % Soluble                                                                     Polypeptide % Intact                                                                              Soluble                                     Formulation.sup.a                                                                           Recovered.sup.b                                                                           Dimer.sup.c                                                                           Dimer.sup.d                                 ______________________________________                                        0.01% Tween ® 20*                                                                       36.1        49.0    11.1                                        0.01% Tween ® 20*                                                                       59.0        69.0    25.6                                        62 mg/ml Mannitol                                                             5 mg/ml Mannitol                                                                            58.3        72.7    56.8                                        50 mg/ml Mannitol                                                                           62.9        83.4    70.3                                        5 mg/ml Trehalose                                                                           117         34.2    53.6                                        50 mg/ml Trehalose                                                                          75.6        61.3    62.1                                        1% CMC        78.2        62.5    65.5                                        No excipient  51.6        6.0     7.9                                         ______________________________________                                         .sup.a All solutions with excipient contained 134 mg/ml hIFNγ,10 mM     sodium succinate, pH 5; "no excipient" formulation contained 256.3 mg/ml      protein, 10 mM sodium succinate, pH 5.0.                                      .sup.b Polypeptide extracted out of methylene chloride (fraction of total     as determined by absorbance at 280 nm.                                        .sup.c Amount of intact dimer measured by SECHPLC native method. All          formulations yielded >99% monomer when assayed by the SECHPLC SDS method.     .sup.d Soluble dimer concentration (mg/ml) based on the amount of soluble     polypeptide recovered and the fraction of dimer (native SECHPLC method).      *Polypeptide concentration in these formulations was 62.8 mg/ml.         

EXAMPLE II Stabilization of Dry and Aqueous Formulations forEncapsulation

In the development of a long acting formulation for recombinant humangrowth hormone the use of a biodegradable polymeric matrix for sustainedrelease of hGH was investigated. The polymer used for this applicationwas a copolymer of lactic and glycolic acids which is often referred toas poly(lactic/glycolic acid) or PLGA. To incorporate hGH into thispolymer, the PLGA must be dissolved in a water immiscible solvent. Themost commonly used solvent for dissolution of PLGA has been methylenechloride which provides both water immiscibility and PLGA solubility.

In general, for production of hGH-PLGA microspheres, the polypeptide wasadded to a solution of methylene chloride containing PLGA. In initialstudies, the polypeptide was added in the form of a milled lyophilizedpowder. After polypeptide addition, the methylene chloride solution wasthen briefly homogenized and the solution was added to an emulsificationbath. This process resulted in the extraction of methylene chloride withthe concomitant formation of PLGA microspheres containing hGH. Thepolypeptide released from these microspheres was then studied todetermine the integrity of hGH after incorporation into themicrospheres. Assessment of released hGH was performed by analyticalsize exclusion chromatography (SEC-HPLC) as well as other techniques.Size exclusion chromatography indicated that hGH was released from thePLGA microspheres in the form of the native monomer, aggregates, and anunknown structure which eluted between the monomer and dimer. Theunknown polypeptide structure has been extensively studied and has beenshown to be a conformational variant of hGH. In addition, the sameaggregates and conformational variant can be obtained by treatment ofhGH with methylene chloride. Thus, the use of methylene chloride in theprocess may cause denaturation and aggregation of hGH.

The release of monomeric native hGH from the PLGA microspheres isrequired for a successful long acting formulation. Previous studiesinvestigated several organic solvents as alternatives to methylenechloride. This research indicated that hGH was susceptible to damage byseveral organic solvents. Since methylene chloride provided the desiredsolvent properties (i.e. water immiscibility, PLGA dissolution, etc.)for PLGA microsphere production and other solvents did not significantlyimprove hGH stability, methylene chloride was chosen for the productionof the PLGA microspheres. The polypeptide used for the solvent study andin the PLGA production process was formulated and lyophilized inammonium bicarbonate buffer at pH 7. Therefore, this study was performedto develop formulations which would stabilize hGH during the productionof the PLGA microspheres.

A. Methods

1. Preparation of hGH Formulations

For development of a methylene chloride stable formulation, hGHlyophilized in ammonium bicarbonate was reconstituted in the desiredbuffer and allowed to dissolve. Undissolved polypeptide was removed bycentrifugation at 13,000 rpm for 1 min.

For each lyophilization, indicted below, the hGH concentration was 10mg/ml. The residual moisture of these formulations was not determined,but the same lyophilization cycle was used in each case.

Milling of lyophilized protein was performed with a pressure drivenimpaction mill and resulted in a fine particulate of hGH.

2. Methylene Chloride Testing of hGH Formulations

The effect of methylene chloride on hGH stability was determined byadding hGH to a solution of methylene chloride. For solid hGHconditions, the ratio of polypeptide mass (mg) to volume of organicsolvent (ml) was 40 mg/ml. For the aqueous hGH conditions, 100 μl of hGHin a buffered solution was added to 1.0 ml of methylene chloride toassess the effects of each buffer system on stabilization of hGH inmethylene chloride. After polypeptide addition, the samples weresonicated for 30 seconds in a 47 kHz bath sonicator (Cole Parmer, Model08849-00) to simulate the homogenization step in the microsphereproduction process. If the formulation stabilized hGH againstdenaturation in this test, it was further assessed by homogenization inmethylene chloride. After sonication or homogenization, the polypeptidewas extracted from the methylene chloride by dilution into a 50 foldexcess of 5 mM NaHPO₄, pH 8. The amount and quality of the polypeptideextracted in this step was determined by polypeptide concentrationmeasurements (absorbance at 278 nm) and size exclusion HPLC (SEC-HPLC).The preferred stable formulation was one that yielded the maximumrecovery of monomeric polypeptide without the formation ofconformational variants or aggregates larger than dimers.

B. Results

1. Excipient Studies of hGH Stabilization

Initial studies of hGH lyophilized in ammonium bicarbonate investigatedthe solubility of the polypeptide in different buffers at various pHconditions. From these studies, it was determined that hGH had themaximum stability and solubility in phosphate buffer (5-10 mM) at pH 8,and thus additional studies were performed with hGH in this buffer.

Initial attempts to prevent aggregation of hGH utilized Tween® 80surfactant in the formulation buffer. As shown in Table III, methylenechloride testing of these aqueous formulations indicated that low Tween®surfactant concentrations (0.1% Tween® 80 surfactant) with 10 mg/mlmannitol provided good recovery of soluble monomeric polypeptide.However, the best results in this experiment were obtained for hGH whichwas formulated in 10 mg/ml mannitol without Tween® 80 surfactant (5 mMNaHPO₄, pH 8). Higher concentrations of Tween® surfactant in theformulation buffer resulted in increased aggregation and decreasedrecovery of soluble polypeptide. For each case shown in Table III, theformulations provided greater stabilization of hGH than the milledpolypeptide which was lyophilized in ammonium bicarbonate.

                  TABLE III                                                       ______________________________________                                        Methylene chloride testing                                                    of aqueous hGH formulations                                                                    Soluble Polypeptide                                                 %               (Mass Fraction of Total)                                        Poly-    %        %    %    %     %                                           peptide.sup.b                                                                          Area.sup.c                                                                             Tri- Di-  Inter-                                                                              Mono-                              Formulation.sup.a                                                                      Recovered                                                                              Recovery mer  mer  mediate                                                                             mer                                ______________________________________                                        1% Tween ®                                                                         85.7     90.0     0.5  3.4  1.1   94.9                               80                                                                            0.1% Tween ®                                                                       70.9     98.3     2.0  3.6  1.8   92.6                               80                                                                            1% Tween ®                                                                         65.0     97.8     3.3  3.4  3.4   90.0                               80 10 mg/ml                                                                   Mannitol                                                                      0.1% Tween ®                                                                       70.9     98.3     0.0  2.2  0.0   97.8                               80                                                                            10 mg/ml                                                                      Mannitol                                                                      10 mg/ml PEG                                                                           97.6     101.1    0.0  2.6  0.0   97.4                               (3350 MW)                                                                     10 mg/ml PEG                                                                           76.4     97.7     1.7  2.8  1.6   93.9                               10 mg/ml                                                                      Mannitol                                                                      5 mM NaPO4,                                                                            55.3     99.4     0.0  3.2  0.0   96.8                               pH 8                                                                          5 mM NaPO4,                                                                            91.7     99.8     0.0  1.8  0.0   98.2                               pH 8                                                                          10 mg/ml                                                                      Mannitol                                                                      ______________________________________                                         .sup.a All solutions contain 5 mM NaPO.sub.4, pH 8                            .sup.b Polypeptide extracted out of methylene chloride (fraction of total     as determined by absorbance at 278 nm.                                        .sup.c SECHPLC results for polypeptide extracted into buffer after            methylene chloride treatment                                             

Methylene chloride testing of solid hGH formulations are shown in TableIV. These results indicated that the formulation which best stabilizedthe protein was 5 mM KPO₄, 2.5 mg/ml trehalose.

                  TABLE IV                                                        ______________________________________                                        Methylene chloride testing                                                    of solid rhGH formulations                                                                    Soluble Protein                                                               (Mass Fraction of Total)                                               %        % Area.sup.c                                                                          %                %                                           Protein.sup.b                                                                          Re-     Tri- %     % Inter-                                                                            Mono-                              Formulation.sup.a                                                                      Recovered                                                                              covery  mer  Dimer mediate                                                                             mer                                ______________________________________                                        Milled Solids                                                                 NH.sub.4 CO.sub.3                                                                      44.5     85.4    7.5  5.9   7.3   79.2                               5 mM NaPO.sub.4,                                                                       85.7     100.    0.0  2.1   0.0   97.8                               pH 8                                                                          5 mM NaPO.sub.4,                                                                       87.6     100.    0.0  3.0   0.0   97.0                               pH 8                                                                          10 mg/ml                                                                      Mannitol                                                                      Homogenized Solids.sup.d                                                      5 mM KPO.sub.4,                                                                        97.3     100.    0.0  2.2   0.0   97.8                               pH 8,                                                                         2.5 mg/ml                                                                     Trehalose                                                                     5 mM NaPO.sub.4,                                                                       96.8     100.    0.0  2.0   0.0   98.0                               pH 8                                                                          10 mg/ml                                                                      Mannitol                                                                      0.3 M Na 94.3     100.    0.0  4.2   0.0   95.8                               Succinate,                                                                    10 mg/ml                                                                      Mannitol,                                                                     pH 7                                                                          ______________________________________                                         .sup.a All samples lyophilized at 10 mg/ml rhGH with buffer and excipient     as shown.                                                                     .sup.b Protein extracted out of methylene chloride (fraction of total) as     determined by absorbance at 278 nm.                                           .sup.c SECHPLC results for protein extracted into buffer after methylene      chloride treatment.                                                           .sup.d Solid lyophilized formulations were homogenized in methylene           chloride at 25,000 rpm for 1 min.                                        

Further studies were performed to determine whether a surfactant couldstabilize the methylene chloride-polypeptide interface. Thus, Tween®surfactant was added to the methylene chloride phase and mixed withsolid hGH (KPO₄, pH 8). The addition of Tween® surfactant to themethylene chloride phase did not improve the stability of the solid-hGH(KPO₄, pH 8) as shown in Table V. In addition, the use of thesurfactant, Span® 80 surfactant, in the methylene chloride phase did notimprove the stability of the solid hGH (KPO₄, pH 8). Further attemptswith Tween® surfactant in the methylene chloride phase were unsuccessfulfor the more stable solid hGH formulation (Mannitol, KPO₄, pH 8). Theseresults along with the aqueous studies indicated that Tween® surfactantis preferably not used with these formulations since it promotesaggregation and decreases the solubility of methylene chloride treatedhGH.

                  TABLE V                                                         ______________________________________                                        Effect of Tween ® surfactant in the                                       methylene chloride phase on solid hGH stability                                               Soluble Polypeptide                                                           (Mass Fraction of Total)                                             %         %        %          %     %                                  Tween ®                                                                          Polypeptide.sup.a                                                                       Area.sup.b                                                                             Tri- %     Inter-                                                                              Mono-                              in MeCl.sub.2                                                                        Recovered Recovery mer  Dimer mediate                                                                             mer                                ______________________________________                                        0.01%  40.8      98.7     5.2  13.0  0.0   81.8                               Tween ®                                                                   80                                                                            0.1%   40.8      102.9    8.0  14.0  0.0   77.9                               Tween ®                                                                   80                                                                            1%     53.8      97.3     7.0  11.6  0.0   81.4                               Tween ®                                                                   80                                                                            ______________________________________                                         .sup.a Polypeptide extracted out of methylene chloride (fraction of total     as determined by absorbance at 278 nm.                                        .sup.b SECHPLC results for polypeptide extracted into buffer after            methylene chloride treatment                                             

To increase the amount of polypeptide loaded into the microspheres, theamount of excipient should be minimized. Therefore, lower concentrationsof mannitol (2 and 5 mg/ml) with 10 mg/ml hGH were used in theformulation buffer (10 mM NaHPO₄, pH 8) and the aqueous solutions weretested for methylene chloride stability. These mannitol concentrationsyielded 20% less soluble monomer than the 10 mg/ml mannitol formulation.Significant reductions in the mannitol concentration would sacrifice thequality of the released polypeptide. Alternative excipients at lowerconcentrations were also attempted. Carboxymethylcellulose (CMC) at 0.5,2, and 5 mg/ml was used in the aqueous formulation (10 mg/ml hGH, 10 nMNaHPO₄, pH 8). CMC at 0.5 mg/ml provided the same fraction of solublemonomer as the 10 mg/ml mannitol formulation, but the amount ofpolypeptide recovered in the aqueous phase was 15% lower. Equal massmixtures of CMC and mannitol (1 mg/ml and 2.5 mg/ml of each) were alsoattempted to provide stability at lower excipient concentrations. Theuse of 2.5 mg/ml of each excipient provided comparable results to the 10mg/ml mannitol formulation. The 0.5 mg/ml CMC and 2.5 mg/ml each of CMCand mannitol formulations were therefore lyophilized to assess their usefor microencapsulation.

To assess formulations for use in the aqueous form, each lyophilizedmaterial was reconstituted to the maximum solubility which was definedas the polypeptide concentration where additional polypeptide would notdissolve in the solution. The maximum concentration of hGH in thisexperiment was achieved with the formulation lyophilized in 10 mg/mlmannitol. This formulation was successfully reconstituted with 5 mMNaHPO₄ buffer, pH 8 to 200 mg/ml of hGH (200 mg/ml mannitol, 100 mMKPO₄) without precipitation of the polypeptide. The formulation withoutexcipients (KPO₄, pH 8) provided the second best solubility at 165 mg/mlof hGH. However, attempts to reconstitute the CMC and CMC/mannitolformulations at high polypeptide concentrations were not successful. Inboth cases, the formulation formed a paste at concentrations greaterthan 100 mg/ml. Methylene chloride testing of the pastes formed from theCMC and CMC/mannitol formulations revealed that the amount ofpolypeptide recovered was significantly reduced (less than 75% recovery)compared to the mannitol formulation, but the soluble fraction wasgreater than 95% monomer. Since a gel-like formulation may have utilityfor stabilizing the inner aqueous phase in the process, anotherthickening agent, gelatin was also attempted. To maintain a lowexcipient concentration while still obtaining a gel for the finalformulation (200 mg/ml hGH), the gelatin formulation was tested at 0.5mg/ml gelatin, 10 mg/ml hGH, 10 mM KPO₄, pH 8. Methylene chloridetesting of this formulation yielded recovery of soluble monomer whichwas comparable to the 10 mg/ml mannitol formulation. Therefore, thisformulation was also lyophilized for further analysis. Reconstitution ofthe lyophilized polypeptide at 200 mg/ml hGH (10 mg/ml gelatin, 100 mMKPO₄, pH 8) resulted in the formation of a paste which had propertiessimilar to those of the CMC/mannitol and CMC formulations at the samehGH concentration.

EXAMPLE III Stability of rhGH Formulations in Ethyl Acetate

Microencapsulation of proteins in biodegradable polymers often requiresthe use of organic solvents to solubilize the polymer. The polymer,typically PLGA, polylactide (PLA), or polyglycolide (PGA), is firstdissolved in an organic solvent that is not completely miscible withwater. The common organic solvents used in this process are methylenechloride and ethyl acetate. These two solvents have very differentphysical and chemical properties. Therefore, it was necessary to assessthe stability of rhGH formulations in both solvents.

The testing of rhGH formulations for stability in ethyl acetate wasperformed by a method similar to the one used for the methylene chloridestudies in the examples above. Solutions of rhGH at 10 mg/ml wereprepared by adding lyophilized solid rhGH (ammonium bicarbonateformulation) to each formulation. As shown in Table VI, the formulationswere prepared with 5 mM KPO₄, pH 8 and contained different excipients,PEG (3350 MW), mannitol, trehalose, and Tween® 20, or combinations ofexcipients. Each rhGH formulation (100 uL) was added to 1 mL of ethylacetate and sonicated for 30 sec to form an emulsion. This emulsion wasthen mixed with 10 mL of 5 mM KPO₄, pH 8 resulting in an overalldilution of rhGH by 100 fold. The rhGH extracted into the buffer wasanalyzed by size exclusion HPLC. Several formulations yielded greaterthan 100% recovery of soluble protein indicating that the amount ofprotein added to the emulsion was greater than the estimated amount (0.1mL×10 mg/mL=1 mg) as the result of the accuracy in volume measurements.In addition, the recovery of soluble protein and the amount of monomerrecovered were generally greater than the rhGH in the same formulationtreated with methylene chloride. Overall, the recovery of solubleprotein was greatest for trehalose (1 & 2 mg/mL), trehalose with PEG (10mg/mL each), mannitol with PEG (10 mg/mL each) and mannitol with Tween®20 (10 mg/mL each). However, only the trehalose (1 & 2 mg/ml) and themannitol with Tween® 20 (10 mg/mL each) also had a high monomer content(greater then 97%). The mannitol/Tween® 20 formulation does not allowadequate solubility for a double emulsion microencapsulation process andit requires a 4:1 excipient to protein ratio (by mass). Thus, theoptimum formulation in these experiments was the 1 mg/mL trehaloseformulation (1:10 excipient to protein ratio and high rhGH solubility).

                  TABLE VI                                                        ______________________________________                                        Ethyl acetate testing of aqueous rhGH                                         formulations as described in the text.                                                          Soluble Protein                                                               (Mass Fraction of Total)                                                   %        %                                                                    Recovery.sup.b                                                                         Large   %     %                                       Formulation.sup.a                                                                            Soluble  Aggreg. Dimer Monomer                                 ______________________________________                                        No excipient   98.9     2.3     3.2   94.5                                    10 mg/mL PEG (3350                                                                           99.8     2.7     2.3   94.9                                    MW)                                                                           5 mg/mL PEG    108.5    1.7     3.0   95.2                                    2 mg/mL PEG    107.2    1.8     3.8   94.3                                    10 mg/mL Mannitol                                                                            96.6     1.7     3.6   94.7                                    2 mg/mL Mannitol                                                                             86.3     4.1     3.8   92.2                                    10 mg/mL Trehalose                                                                           100.1    1.8     4.5   93.7                                    2 mg/mL Trehalose                                                                            119.8    0.4     2.0   97.7                                    1 mg/mL Trehalose                                                                            111.1    0.6     2.3   97.1                                    10 mg/mL PEG (3350                                                                           115.6    3.8     2.9   93.3                                    MW) 10 mg/mL Trehalose                                                        2 mg/mL PEG (3350                                                                            93.0     0.8     3.1   96.1                                    MW) 2 mg/mL Trehalose                                                         1 mg/mL PEG (3350 MW)                                                                        95.8     4.5     3.3   92.2                                    1 mg/mL Trehalose                                                             10 mg/mL PEG (3350 MW)                                                                       116.3    1.2     2.5   96.3                                    10 mg/mL Mannitol                                                             2 mg/mL PEG (3350 MW)                                                                        106.5    1.7     2.7   95.6                                    2 mg/mL Mannitol                                                              0.1% Tween ® 20                                                                          122.8    0.8     1.6   97.6                                    10 mg/mL Mannitol                                                             ______________________________________                                         .sup.a All initial test solutions contained 10 mg/mL rhGH and 5 mM            KPO.sub.4, pH 8 except three of the formulations which were at rhGH           concentrations less than 10 mg/mL (no excipient: 9.39 mg/mL; 10 mg/mL         mannitol/10 mg/mL PEG: 7.84 mg/mL; 10 mg/mL mannitol: 9.71 mg/mL).            .sup.b SECHPLC results for protein extracted into buffer after ethyl          acetate treatment. The percent recovery of soluble protein was defined as     the ratio of the concentrations from the total peak area of the sample an     the appropriate controls (same formulation) times 100%. The control rhGH      concentration was determined by absorbance at 278 nm and the sample rhGH      concentration was calculated as a 100 fold dilution of the stock material     based on the dilutions used in the overall method (0.1 mL in 1 mL EtAc        added to 10 mL buffer).                                                  

EXAMPLE IV Stability of Spray Dried rhGH Formulations in OrganicSolvents

The double emulsion technique (water-in-oil-in-water) formicroencapsulation can only provide moderate loading of drug in thefinal product. The drug loading is limited by the solubility of the drugin water and the volume of aqueous drug that can be added to the polymerin organic solvent. Volumes of greater than 0.5 mL of drug per gram ofpolymer typically result in a large initial burst of drug from themicrospheres. To avoid these difficulties, a solid drug formulation canbe used in place of the aqueous drug solution. Thus, asolid-in-oil-in-water process can be used to produce microspheres withhigh drug loading (greater then 10%) with low to moderate initialbursts.

The solid drug formulation used for microencapsulation must be stable inorganic solvents and it must have a small size (1-5 μm) relative to themicrospheres (30-100 μm) to permit high loading and low burst of thedrug. For protein formulations, one method of obtaining small driedsolids is spray drying. A recent report by Mummenthaler et al., Pharm.Res. 11(1):12-20 (1994) describes the process of spray drying rhGHformulations. Since rhGH is easily denatured by surface interactionssuch as air-liquid interfaces, the spray drying of rhGH must beperformed with surfactants in the rhGH formulation. However, as notedabove, the presence of some surfactants can have a negative effect onthe stability of rhGH in methylene chloride. Spray dried rhGHformulations with different surfactants and trehalose, which were aboveobserved to be the best for stabilization of the aqueous rhGHformulations, were tested for stability in methylene chloride and ethylacetate.

Spray dried rhGH was prepared from each of the formulations listed inTable VII. These formulations were sprayed at 5 mL/min with an inlettemperature of 90° C., an air nozzle flow rate of 600 L/hr, and a dryingair rate of 36,000 L/hr. The spray dried rhGH was then collected fromthe filter and the cyclone units of the spray drier. The final solidusually was approximately 5 μm in diameter.

The spray dried rhGH powder was then tested for stability by treatmentwith either ethyl actate or methylene chloride. A spray dried powdermass equivalent to 10 mg of rhGH was added to 2 mL of the organicsolvent in a 3 cc glass test tube. The suspension was next homogenizedat 10,000 rpm for 30 sec with a microfine homogenization tip. Aftermixing, 20 μL of the homogeneous suspension was added to 980 μL of 5 mMKPO₄, pH 8 to extract out the protein. The extracted proteinconcentration was determined by absorbance at 278 nm and the sample wasalso analyzed by size exclusion chromatography. As shown in Table VII,the formulation without surfactant had the greatest extent ofaggregation when treated with methylene chloride. This aggregation waslikely the result of the surface denaturation of rhGH during the dryingprocess as previously observed for spray drying of rhGH. By addingeither Tween® 20 or PEG (3350 MW) to the formulation, the amount ofaggregation for the methylene chloride treated samples was reduced, butthe overall recovery yield was still low and the monomer content wasmuch less than 90%. In contrast, if the same spray dried rhGHformulations containing surfactant were treated with ethyl acetate, theamount of aggregation was neglible and complete recovery of monomericrhGH was achieved. Therefore, spray dried rhGH formulations consistingof trehalose and either Tween® 20 or PEG (3350 MW) were stable in ethylacetate but did not protect the protein from denaturation in methylenechloride.

                  TABLE VII                                                       ______________________________________                                        Stability of spray dried solid rhGH formulations in                           methylene chloride and ethyl acetate.                                                             Soluble Protein                                                       %        %        %     %    %                                                Recovery.sup.a                                                                         Recovery.sup.b                                                                         Large Di-  Mono-                                Formulation (Total)  (Soluble)                                                                              Aggreg                                                                              mers mer                                  ______________________________________                                        Methylene Chloride Tests                                                      15 mg/mL rhGH                                                                             --       --       12.3  8.8  75.4                                 3.75 mg/mL trehalose                                                          10 mg/ml rhGH                                                                             56.5     65.2     1.9   13.3 78.1                                 2.5 mg/mL trehalose                                                           0.2% Tween ® 20                                                           10 mg/mL rhGH                                                                             50.7     56.7     3.9   12.3 77.7                                 2.5 mg/mL trehalose                                                           0.2% PEG (3350 MW)                                                            Ethyl Acetate Tests                                                           10 mg/mL rhGH                                                                             111.7    126.8    0.9   0.0  99.2                                 2.5 mg/mL trehalose                                                           0.2% Tween ® 20                                                           10 mg/mL rhGH                                                                             114.3    125.5    1.1   0.0  98.9                                 2.5 mg/mL trehalose                                                           0.2% PEG (3350 MW)                                                            5.0 mg/mL rhGH                                                                            106.8    110.4    0.3   3.0  96.7                                 1.25 mg/mL trehalose                                                          0.2% Tween ® 20                                                           ______________________________________                                         .sup.a The total recovery of protein was defined as the amount of protein     extracted into buffer after treatment in the organic solvent divided by       the calculated amount of protein added to the extraction buffer (0.02 mL      × 5 mg/mL).                                                             .sup.b SECHPLC results for protein extracted into buffer after treatment      with organic solvent. The percent recovery of soluble protein was defined     as the ratio of the concentrations from the total peak area of the sample     and a reference standard times 100%. The control and sample rhGH              concentrations were determined by absorbance at 278 nm.                  

EXAMPLE V Stability of Spray Freeze-Dried rhGH Formulations in MethyleneChloride and Ethyl Acetate

Spray drying at high temperatures can have a detrimental effect on theprotein and it produces protein particles which are often hollow spheres(Mummenthaler et al., Pharm. Res. 11(1):12-20 (1994). In addition, it isdifficult to collect the small particles 1-5 μm) required formicroencapsulation and the overall yield of these particles is usuallyvery low (less than 50%). An alternative to high temperature spraydrying is spray freeze-drying. Spray freeze-drying of rhGH formulationsresults in a fine particles (2-3 μm) that readily break apart into verysmall solids (less than 1 μm). This type of solid formulation ispreferred for microencapsulation in a polymer matrix since it canprovide a high loading (able to pack more solid into 30-100 μmmicrospheres) of homogeneously dispersed solid protein (reduced burstdue to fine suspension).

Spray freeze-drying of rhGH was performed with the formulations listedin Table VIII. Again, a surfactant was required to stabilize rhGH duringthe spraying process but other proteins which are not easily denaturedby surface interactions would probably not require the use of asurfactant. The spray freeze-dried rhGH was prepared by pumping theformulation at 5 mL/min and operating the air nozzle at 600 L/hr as usedfor high temperature spray drying (Mummenthaler et al., Pharm. Res.11(1):12-20 1994). The solutions were sprayed into an open metal tray ofliquid nitrogen. After spraying, the tray was placed in a prechilledlyophilizer set at -30° C. The liquid nitrogen was allowed to evaporateand the protein was then lyophilized (primary drying: -30° C., 100mTorr, 52 hrs; secondary drying: 5° C., 100 mTorr, 18 hrs). The finalpowder was then removed and placed in sealed glass vials prior to use.

The spray freeze-dried rhGH powder was then tested for stability bytreatment with ethyl actate and methylene chloride. A spray freeze-driedpowder mass equivalent to 10 mg of rhGH was added to 2 mL of the organicsolvent in a 3 cc glass test tube. The suspension was next homogenizedat 10,000 rpm for 30 sec with a microfine homogenization tip. Aftermixing, 20 μL of the homogeneous suspension was added to 980 μL of 5 mMKPO₄, pH 8 to extract out the protein. The extracted proteinconcentration was determined by absorbance at 278 nm and the sample wasalso analyzed by size exclusion chromatography. As shown in Table VIII,the spray freeze-dried formulation containing PEG was more stable inmethylene chloride than the formulation containing Tween® 20 as observedabove with the aqueous formulations. However, both formulations did notyield high recovery of monomeric rhGH. When these same formulations weretreated with ethyl acetate, complete recovery of monomeric protein wasachieved with both formulations. The trehalose in the formulationsprovided stabilization against organic solvent denaturation (ethylacetate) while the surfactants stabilized the protein against surfacedenaturation during spray freeze-drying. Thus, spray freeze driedformulations containing both trehalose and a surfactant will yieldcomplete recovery of rhGH from ethyl acetate.

                  TABLE VIII                                                      ______________________________________                                        Stability of spray freeze-dried solid rhGH                                    formulations in methylene chloride and ethyl                                  acetate.                                                                                          Soluble Protein                                                       %        %        %     %    %                                                Recovery.sup.a                                                                         Recovery.sup.b                                                                         Large Di-  Mono-                                Formulation (Total)  (Soluble)                                                                              Aggreg                                                                              mers mer                                  ______________________________________                                        Methylene Chloride Tests                                                      5 mg/mL rhGH                                                                              37.2     34.0     6.2   8.3  85.5                                 1.25 mg/mL trehalose                                                          0.2% Tween ® 20                                                           5 mg/ml rhGH                                                                              68.8     66.8     2.3   15.8 78.8                                 1.25 mg/mL trehalose                                                          0.2% PEG (3350 MW)                                                            Ethyl Acetate Tests                                                           5 mg/mL rhGH                                                                              94.6     117.7    0.5   0.9  98.7                                 1.25 mg/mL trehalose                                                          0.2% Tween ® 20                                                           5 mg/ml rhGH                                                                              97.7     104.7    0.6   0.0  99.4                                 1.25 mg/mL trehalose                                                          0.2% PEG (3350 MW)                                                            ______________________________________                                         .sup.a The total recovery of protein was defined as the amount of protein     extracted into buffer after treatment in the organic solvent divided by       the calculated amount of protein added to the extraction buffer (0.02 mL      × 5 mg/mL).                                                             .sup.b SECHPLC results for protein extracted into buffer after treatment      with organic solvent. The percent recovery of soluble protein was defined     as the ratio of the concentrations from the total peak area of the sample     and a reference standard times 100%. Control and sample rhGH                  concentrations were determined by absorbence at 278 nm.                  

What is claimed is:
 1. A composition for controlled release of apolypeptide, the composition comprising a polypeptide admixed with anexcipient, an organic solvent, and a polymer matrix, wherein theexcipient is trehalose.
 2. The composition of claim 1 wherein thepolypeptide admixed with the excipient is in an aqueous formulation. 3.The composition of claim 1 wherein the polypeptide admixed with theexcipient is dry.
 4. The composition of claim 1 wherein the polypeptideadmixed with the excipient is lyophilized.
 5. The composition of claim1, wherein the polymer is a polylactide.
 6. The composition of claim 1,further comprising a buffer.
 7. The composition of claim 6, furthercomprising a preservative.